EP0632829A1 - Antagonistes de recepteur de thrombine - Google Patents

Antagonistes de recepteur de thrombine

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Publication number
EP0632829A1
EP0632829A1 EP93907162A EP93907162A EP0632829A1 EP 0632829 A1 EP0632829 A1 EP 0632829A1 EP 93907162 A EP93907162 A EP 93907162A EP 93907162 A EP93907162 A EP 93907162A EP 0632829 A1 EP0632829 A1 EP 0632829A1
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EP
European Patent Office
Prior art keywords
arg
thrombin
trip
val
lys
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EP93907162A
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German (de)
English (en)
Inventor
John M. Maraganore
Andrew L. Frelinger, Iii
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Biogen Inc
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Biogen Inc
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Publication of EP0632829A1 publication Critical patent/EP0632829A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6429—Thrombin (3.4.21.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21005—Thrombin (3.4.21.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

  • This invention relates to novel, biologically active molecules which bind to and inhibit the thrombin receptor.
  • the molecules of this invention are characterized by a portion which binds to the "hirudin- like" domain of the thrombin receptor (amino acids 52-69) .
  • These thrombin receptor antagonists are further characterized by their ability to inhibit thrombin-induced platelet aggregation without affecting the catalytic activity of thrombin.
  • the thrombin receptor antagonist of this invention comprises the formula: Y-A,-W-X,-X 2 -X 3 -X 4 -A 2 -Z; wherein Y is hydrogen, a peptide of from 1 to 10 amino acids, either the same or different, or an acyl or alkyl chain comprising from 1 to 30 backbone atoms; each of A,and A 2 , either the same or different, are selected from the group consisting of a positively charged amino acid and an acyl or alkyl chain comprising from 1 to 10 backbone atoms and a positively charged side group; and each X is any amino acid, either the same or different; and Z is OH, a peptide of from 1 to 11 amino acids, either the same or different, or an acyl or alkyl chain comprising from 1 to 33 backbone atoms, and wherein said antagonist is optionally cyclized by a covalent bond between Y and Z; with the proviso that at least one of W, X, and X
  • Thrombin is a naturally occurring protein which has several bioregulatory roles [J. . Fenton, II, "Thrombin Bioregulatory Functions", Adv. Clin. Enzymol.. 6, pp. 186-93 (1988)].
  • thrombin In addition to being intimately involved in blood coagulation, thrombin is also known to play a role in platelet and endothelial cell activation, smooth muscle cell and neuroblast proliferation and bone resorption.
  • Thrombin exerts its various biological effects through one of two mechanisms. The first is purely enzymatic, involving only the catalytic activity of thrombin. This is the mechanism by which thrombin converts fibrinogen to fibrin, the final step in the coagulation cascade.
  • thrombin activates cells via its receptor by first associating with the hirudin-like extracellular domain of the receptor (receptor amino acids 52-69) [L.-W. Liu et al., "The Region of the receptor
  • Thrombin Receptor Resembling Hirudin Binds to Thrombin and Alters Enzyme Specificity J. Biol. Chem., 266,. pp. 16977-80 (1991)].
  • Thrombin then cleaves off a 41 amino acid peptide from its receptor's extracellularly located N-terminus, exposing a new N-terminus on the receptor. The newly exposed N-terminus then interacts with another, distant part of the receptor, resulting in activation. It is believed that activation may ultimately involve a secondary messenger, such as a kinase or cAMP, which is triggered by the cleaved receptor.
  • a secondary messenger such as a kinase or cAMP
  • the tethered ligand peptide can activate the thrombin receptor in the absence of thrombin. This activation does not require the presence of an extracellular anionic domain in the receptor [T.-K. H. Vu et al. , supra1.
  • the receptor-driven mechanism is responsible for thrombin-induced platelet aggregation and release reactions. Such reactions initiate arterial clot formation. In addition, the receptor mechanism is thought to be responsible for thrombin-induced endothelial cell activation. This activation stimulates the synthesis of platelet activating factor (PAF) in these cells [S. Prescott et al., "Human Endothelial Cells in Culture Produce Platelet- Activating Factor (l-alkyl-2-acetyl-sn-glycero-3- phosphocholine) When Stimulated With Thrombin", Proc. Natl. Acad. Sci. USA. 81, pp. 3534-38 (1984)].
  • PAF platelet activating factor
  • thrombin may promote inflammation by altering endothelial cells to produce increased vascular permeability which can lead to edema [P. J. Del Vecchio et al., "Endothelial
  • Smooth muscle cells are also known to express thrombin receptors on their surface [D. T. Hung et al., "Thrombin-induced Events in Non-Platelet Cells Are Mediated by the Unique Proteolytic Mechanism Established for the Cloned Platelet Thrombin Receptor, J. Cell Biol.. 116, pp. 827-32 (1992)].
  • thrombin interacts with these cells, a proliferative response is generated.
  • This proliferation of smooth muscle cells may contribute to restenosis following balloon angioplasty and subsequent coronary failure [A. J. B. Brady et al., "Angioplasty and Restenosis", Brit. Med. J.. 303, pp. 729-30 (1991)].
  • Other cells which have thrombin receptors are fibroblasts, neuroblasts and osteoclasts. For each of these cells, thrombin has been shown to be responsible for some bioregulatory function.
  • thrombin has been implicated in neurodegenerative diseases because of its ability to cause neurite retraction [D. Gurwitz et al. , "Thrombin Modulates and Reverses Neuroblasto a Neurite Outgrowth", Proc. Natl. Acad. Sci. USA. 85, pp. 3440-44 (1988) ] . And thrombin has been hypothesized to play a role in osteoporosis due to its ability to stimulate bone resorption by osteoclasts [D. N. Tatakis et al., "Thrombin Effects On Osteoblastic Cells -II. Structure- Function Relationships", Biochem. Biophys. Res. Comm.. 174, pp.
  • thrombin Compounds which inhibit thrombin directly may inhibit some or all of thrombin's functions to one degree or another. All active site inhibitors of thrombin inhibit both enzymatic and receptor-mediated functions of the molecule. Surprisingly, other thrombin inhibitors which do not bind to the active site can also inhibit both mechanisms. For example, peptides modelled on the C-terminal amino acid sequence of hirudin, a naturally occurring anticoagulant isolated from leeches, inhibit both fibrinogen cleavage, as well as thrombin-induced platelet and endothelial cell activation [copending United States patent application Serial No. 677,609].
  • thrombin Other compounds are capable of selectively inhibiting thrombin's activities, but these compounds do not inhibit thrombin or its receptor directly and therefore are less specific.
  • heparin which complexes with and activates antithrombin III, affects fibrin clot formation without affecting platelet-dependent thrombosis. But certain patients cannot be treated with heparin because of circulating antibodies against this compound. Also, heparin is known to cause bleeding complications.
  • Arg-Gly-Asp- containing peptides inhibit platelet activation, without inhibiting fibrin formation. However, these peptides are general antiplatelet agents and are not specific for thrombin-induced platelet activation.
  • thrombin's functions work by competitively binding to the platelet surface protein, glycoprotein lib/Ilia.
  • a direct and selective antagonist of thrombin's functions would have great utility.
  • Such a compound would be particularly useful in preventing restenosis following angioplasty — a phenomenon linked to thrombin-induced smooth muscle cell proliferation [J. N. Wilcox, "Thrombin and Other Potential Mechanisms Underlying Restenosis", Circulation f 84, pp. 432-35 (1991)].
  • Thrombin receptor antagonists would also be useful in treating or preventing arterial thrombosis without blocking fibrin formation.
  • the present invention solves the problems set forth above by providing novel molecules which competitively bind to and inhibit the thrombin receptor without affecting thrombin's activities toward fibrinogen and other proteins involved in coagulation.
  • novel antagonists are characterized by a portion which binds to the "hirudin-like" anionic domain of the thrombin receptor (amino acids 52-69) .
  • the antagonists of this invention are preferably modelled on the anion binding exosite portion of thrombin (amino acids 65-83) , or a portion thereof, which is known to associate with the hirudin-like domain of its receptor.
  • the thrombin receptor antagonists of this invention may be utilized in compositions and methods for controlling certain thrombin-mediated functions without the risk of bleeding.
  • compositions comprising the antagonists of this invention are useful in inhibiting platelets; treating or preventing inflammation; treating or preventing bone resorption (a process prevalent in osteoporosis) ; treating or preventing neurodegenerative disease; and inhibiting smooth muscle cell proliferation. This last utility is particularly important following coronary angioplasty, where restenosis, which is believed to be caused by smooth muscle cell growth at the initial vessel blockage site, occurs with relatively high frequency.
  • the antagonist molecules of the present invention may be prepared by chemical synthesis techniques, commercially feasible amounts may be produced inexpensively. Moreover, because the antagonist molecules of the present invention are significantly smaller than the thrombin inhibitors presently employed in medical treatment, they are less likely to stimulate an undesirable immune response in patients treated with them.
  • Figure 1 depicts the inhibition of platelet aggregation by varying concentrations of TRIP-1 in the presence of varying concentrations of thrombin.
  • Figure 2 depicts the lack of antiplatelet effect of TRIP-1 on platelets treated with the tethered ligand peptide of the thrombin receptor.
  • Figure 3 depicts the efficacy of TRIP-1 in the inhibition of platelet aggregation induced by various agonists.
  • Figure 4 depicts the comparative efficacies of TRIP-1, TRIP-4 (both linear and cyclized) and TRIPr5 (both linear and cyclized) in inhibiting thrombin- induced platelet aggregation.
  • Figure 5 depicts the relative thrombin receptor inhibitory activity of single position alanine and glycine substitutions in TRIP-6 compared to the unsubstituted antagonist.
  • Figure 6 depicts the inhibition of thrombin- induced smooth muscle cell proliferation by TRIP-6 and TRIP-20, as measured by 3 H-thymidine uptake.
  • Figure 7 depicts the nucleotide sequence of a plasmid which encodes an erabutoxinA-TRIP fusion protein.
  • Figure 8 depicts the ability of various erabutoxinA-TRIP fusion proteins expressed in phage to bind to an immobilized thrombin receptor peptide.
  • Leu - leucine lie - isoleucine
  • any amino acid as used herein includes the L-isomers of the naturally occurring amino acids, as well as other "non-protein" ⁇ -amino acids commonly utilized by those in the peptide chemistry arts when preparing synthetic analogues of naturally occurring peptides.
  • the naturally occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, 7-carboxyglutamic acid, arginine, ornithine and lysine.
  • non-protein ⁇ -amino acids include norleucine, norvaline, alloisoleucine, homoarginine, thioproline, dehydroproline, hydroxyproline (Hyp) , isonipecotic acid (Inp) , homoserine, cyclohexylglycine (Chg) , ⁇ -amino-n-butyric acid (Aba) , cyclohexylalanine (Cha) , aminophenylbutyric acid (Pba) , phenylalanines substituted at the ortho, meta, or para position of the phenyl moiety with one or two of the following: a (C,-C 4 ) alkyl, a (Cj-Q,) alkoxy, halogen or nitro groups or substituted with a methylenedioxy group; ⁇ -2- and 3-thienylal-alanine, ⁇ -2- and 3-furanylalanine, ⁇ -2-
  • amino acids referred to in this application are in the L-form.
  • positively charged amino acid includes any naturally occurring or non-naturally occurring amino acid having a positively charged side chain. Examples of positively charged amino acids are arginine, lysine, histidine, homoarginine, ornithine and delta-alkyl ornithine.
  • amino acid containing an aryl side chain as used herein means any amino acid having an aromatic group. Tyrosine, phenylalanine, tryptophan, O-sulfate esters of tyrosine and 5-nitrotyrosine exemplify such amino acids.
  • polar amino acid means any amino acid having an uncharged side chain which is relatively soluble in water. Examples include glutamine, asparagine, glycine, serine, hydroxyproline and homoserine.
  • hydrophobic amino acid means any amino acid having an uncharged side chain which is relatively insoluble in water. This group includes leucine, valine, tryptophan, norleucine, norvaline, alloisoleucine, thioproline, dehydroproline, cyclohexylalanine and cyclohexylglycine.
  • acidic amino acid refers to any amino acid having a negatively charged side chain. Examples include aspartic acid and glutamic acid.
  • patient as used in this application refers to any mammal, especially humans.
  • backbone chain refers to the portion of a chemical structure that defines the smallest number of consecutive bonds that can be traced from one end of that chemical structure to the other.
  • the atomic components that make up a backbone chain may comprise any atoms that are capable of forming bonds with at least two other atoms.
  • each of the following chemical structures is characterized by a backbone chain of 7 atoms (the atoms which comprise the backbone chain are indicated in boldface) :
  • the present invention relates to molecules that compete with thrombin for binding to its cell surface receptor. This property allows the molecules of this invention to inhibit those functions of thrombin that are mediated through its receptor.
  • the thrombin receptor antagonists of the present invention are characterized by a region that binds to the "hirudin- like" domain of the thrombin receptor.
  • that region is modelled on the thrombin anion binding exosite (thrombin amino acids 65-83; SEQ ID NO:l: Leu- Val-Arg-Ile-Gly-Lys-His-Ser-Arg-Thr-Arg-Tyr-Glu-Arg- Asn-Ile-Glu-Lys-Ile-Ser) , or a portion thereof.
  • the antagonists of this invention are further characterized by: (1) the ability to inhibit thrombin-induced platelet aggregation in a simple m vitro assay, and (2) the inability to inhibit thrombin's catalytic activity.
  • the molecule In designing an antagonist molecule according to this invention, two important considerations must be taken into account. First, the molecule must be able to physically associate with the thrombin receptor.
  • the present theory of thrombin receptor activation suggests that the initial step in activation requires, at a minimum, an ionic interaction between the receptor and thrombin.
  • thrombin utilizes its anion binding exosite region, i.e., a cluster of positive charges, to bind to the anionic domain of the receptor. It is also probable that other molecular interactions, such as hydrogen bonding and hydrophobic interactions, are important for this association. Therefore, the identification and maintenance of these interactions are critical in designing a potent thrombin receptor antagonist.
  • the second consideration in designing the antagonists of this invention is secondary and tertiary structure. While certain portions of the antagonist do not directly participate in molecular interactions with the receptor, they may play a role in the overall conformation of the antagonist. This, in turn, can have a dramatic effect on potency. If the antagonist cannot assume the proper conformation, the molecular interactions required for association with the receptor cannot be achieved, even if the components capable of forming such interactions are present in the molecule. Accordingly, an antagonist of this invention must be designed so that it assumes a conformation which allows it to associate with the receptor. Conformational requirements may be in the nature of overall three- dimensional structure and orientation of the antagonist, or merely the spacing between two sites on the antagonist which directly interact with the receptor.
  • thrombin receptor antagonists of this invention In order to identify and define the important structural and conformational attributes necessary to the thrombin receptor antagonists of this invention, one of ordinary skill in the art begins with a peptide consisting of amino acids 65-83 of thrombin. This peptide itself is a thrombin receptor antagonist of the present invention.
  • both an alanine- and a glycine-scanning procedure are carried out.
  • a series of peptides, each having a single alanine or glycine substitution at a different residue, is synthesized.
  • the peptides are then assayed to determine if they inhibit thrombin- induced platelet aggregation, without affecting thrombin catalyzed fibrin formation.
  • an arginine-to-alanine or arginine-to- glycine substitution which resulted in reduced activity suggests the location of an important positive charge - - either an ionic interaction with the receptor or an intramolecular ionic interaction within the antagonist — which is required to maintain optimal conformation.
  • a serine-to-alanine or serine-to-glycine substitution which had a negative effect on activity indicates the location of an important hydrogen bond.
  • the hydrogen bond may be between the antagonist and the thrombin receptor, or it may be an intramolecular hydrogen bond that plays an important role in the conformation of the antagonist.
  • alanine is optically active, it is more bulky and more hydrophobic.
  • aianine- substituted peptide retains its inhibitory activity while the corresponding glycine-substituted peptide does not, one of skill in the art will know that the substituted position requires one of the three above- described characteristics at the substituted position to be active.
  • Deletion peptides from this series which have significantly lower platelet inhibitory activity indicate the location of components which provide critical spacing in the antagonist. This may be verified by replacing the deleted amino acid with a different, yet analogous structure. For example, substitution of any confo ⁇ nationally important amino acid with a three carbon alkyl chain without a significant loss of activity confirms that spacing is critical at that part of the molecule. Additional information about important structural and conformational features necessary for designing a potent thrombin receptor antagonist of this invention may be obtained through 3-dimensional X-ray crystallographic procedures coupled with computer modelling. Specifically, one of ordinary skill in the art may analyze a thrombin receptor/thrombin amino acid 65-83 peptide complex using such a method. Alternatively, one could use the X-ray crystallographic data on the analogous C-terminal hirudin peptide- thrombin complex [E. Skrzypczak-Jankun et al.,
  • thrombin receptor antagonists of this invention may be designed and synthesized. This is achieved by substituting the identified key residues of the thrombin peptide with other components having similar features. These substitutions will initially be conservative, i.e., the replacement component will have approximately the same size, shape, hydrophobicity and charge as the key residue. Those of ordinary skill in the art are well aware of appropriate replacements for a given amino acid [Dayhoff et al., in Atlas of Protein Sequence and Structure No. 5. 1978 and Argos et al., EMBO J. r 8, pp. 779-85 (1989)].
  • Typical conservative substitutions for an amino acid are other amino acids with similar charges. For example, aspartic acid for glutamic acid, arginine for lysine, asparagine for glutamine, hydroxyproline for proline and vice versa. Substitutions with non-natural amino acids may also be performed to reduce the peptidic nature of the antagonist. Some examples are cyclohexylalanine for tyrosine, sarcosine for glycine, statine for threonine and homoarginine for arginine. These modifications may increase the biological stability of the antagonist, in addition to increasing its potency. After the molecule containing the substitute component is shown to be an effective thrombin receptor antagonist, less conservative replacements may be made at the same position.
  • substitutions typically involve the introduction of non-amino acid components which contain the important feature imparted by the amino acid at that position.
  • Such substitutes are well-known in the art.
  • the sequence Leu- Val-Arg (corresponding to amino acids 65-67 of thrombin) can be replaced by p-guanidinobenzoic acid. This substitution maintains the hydrophobicity of Leu- Val, as well as the guanidinium functionality of Arg.
  • novel thrombin receptor antagonists may be designed by insertions at various sites along the thrombin 65-83 peptide.
  • a series of peptides having a single alanine insertion at various sites is synthesized. Those peptides from this series which retain antiplatelet activity indicate potential insertion sites.
  • the insertions may potentially affect the molecular interactions between the antagonist and the thrombin receptor and how they affect conformation of the antagonist. For example, the insertion of an anionic component adjacent to a critical cationic amino acid in thrombin 65-83 could interfere with an important ionic interaction and should therefore be avoided. Similarly, the insertion of a component which is known to cause structural perturbations, e.g., a proline, should also be avoided. Using any or all of the above deletion, substitution and insertion techniques allows those of ordinary skill in the art to design thrombin receptor antagonists according to this invention.
  • the potential effect of any of these changes may be theoretically observed prior to synthesizing the antagonist through the use of computer modelling techniques known in the art.
  • Such modelling allows one to observe the predicted structure of a thrombin receptor complexed with the potential antagonist. If that theoretical structure suggests insufficient interaction between the receptor and the potential antagonist, one need not spend time and resources in synthesizing and testing the molecule.
  • computer modelling indicates a strong interaction, the molecule may then be synthesized and assayed for antiplatelet activity. In this manner, inoperative molecules may be eliminated before they are synthesized.
  • Cyclization may allow the antagonist to assume a more favorable conformation for association with the thrombin receptor. Cyclization may be achieved by methods well-known to those in the . art. One method is the formation of a disulfide bond between two non-adjacent cysteine residues (D- or L- conformation) or any two appropriately spaced components having free sulfhydryl groups. It will be understood that disulfide bonds, as well as other intramolecular covalent bonds, may be formed between a variety of components within the antagonist. The components which form such bonds may be side chains of amino acids, non-amino acid components or a combination of the two.
  • truncated peptides i.e., multiple N- and/or C-terminal deletions
  • truncated peptides i.e., multiple N- and/or C-terminal deletions
  • applicants have identified the minimal portion of thrombin necessary for receptor inhibition — Arg-Ile-Gly-Lys-His-Ser-Arg (thrombin amino acids 67-73; amino acids 3-9 of SEQ ID NO:l).
  • Applicants have made further substitutions within this minimal sequence and have developed peptides which have increased inhibitory activity over that thrombin peptide. These substitutions are discussed below and in more details in the specific examples.
  • a preferred thrombin receptor antagonist of this invention which comprises the formula: Y-A J - -X J ⁇ -X ⁇ A Z -Z; wherein Y is hydrogen, a peptide of from 1 to 10 amino acids, either the same or different, or an acyl or alkyl chain comprising from 1 to 30 backbone atoms; each of A !
  • a 2 are selected from the group consisting of a positively charged amino acid and an acyl or alkyl chain comprising from 1 to 10 backbone atoms and a positively charged side group; W and each X is any amino acid, either the same or different; and Z is OH, a peptide of from 1 to 11 amino acids, either the same or different, or an acyl or alkyl chain comprising from 1 to 33 backbone atoms, and wherein said antagonist is optionally cyclized by a covalent bond between Y and Z; with the proviso that at least one of W, X, and X 2 is not a basic amino acid.
  • More preferred antagonists of the present invention comprise the general formula indicated above, wherein Y has the formula Y,-B n -C m , wherein Yj is hydrogen, a peptide of from l to 6 amino acids, either the same or different, or an acyl or alkyl chain comprising from l to 18 backbone atoms; B is a hydrophobic amino acid; C is an acidic amino acid, either the same or different, n is 0 or 1; and m is an integer from 1 and 3.
  • antagonists wherein A is Arg; antagonists wherein A 2 is Arg; antagonists wherein W is lie, D-Cys or Ala; antagonists wherein X 2 is an anionic amino acid; antagonists wherein X 3 is His; and antagonists wherein
  • X 4 is Ser.
  • the most preferred antagonists of the present invention are referred to as thrombin receptor inhibitory peptides or TRIPS. These antagonists have the formulae:
  • TRIP-1 SEQ ID NO:2: Leu-Val-Arg-Ile-Gly-Lys-His-Ser- Arg-Thr-Arg-Tyr-Glu-Arg-Asn-Ile-Glu;
  • TRIP-2 Leu-Val-Arg-Ile-Cys-Lys-His-Ser-Arg-Thr-Arg- Tyr-Glu-Arg-Asn-Ile-(D-Cys) ;
  • TRIP-3 SEQ ID NO:3: Leu-Cys-Arg-Ile-Gly-Lys-His-Ser- Arg-Thr-Arg-Tyr-Glu-Arg-Asn-Ile; TRIP-4: Leu-Val-Arg-(D-Cys)-Gly-Lys-His-Ser-Arg-Thr- Arg-Tyr-Glu-Arg-Asn-Cys-Glu-Lys-Ile; and
  • TRIP-5 Leu-Val-Arg-(D-Cys)-Gly-Lys-His-Ser-Arg-Thr- Arg-Tyr-Glu-Arg-Asn-(D-Cys)-Glu-Lys-Ile;
  • TRIP-6 SEQ ID NO:4: Leu-Val-Arg-Ile-Gly-Lys-His-Ser- Arg-Thr-Arg-Tyr-Glu-Arg-Asn-Ile-Glu-Lys-Ile;
  • TRIP-7 SEQ ID NO:5: Leu-Val-Arg-Ile-Gly-Orn-His-Ser- Arg-Thr-Arg-Tyr-Glu-Arg-Asn-Ile-Glu-Lys-Ile;
  • TRIP-8 SEQ ID NO:6: Leu-Val-Arg-Ile-Gly-Lys-His-Ser- Arg-Thr-Arg-Tyr-Asp-Arg-Asn-Ile-Glu-Lys-Ile; TRIP-9: SEQ ID NO:7: Leu-Val-HArg-Ile-Gly-Lys-His-
  • TRIP-10 SEQ ID NO:8: Leu-Val-Arg-Ile-Gly-Lys-His-Ser- Arg-Thr-Arg-Tyr-Glu-Arg-Asn-Ile-Glu-Lys-Cha- Gly;
  • TRIP-11 SEQ ID NO:9: MPR-Val-Arg-Ile-Gly-Lys-His-Ser- Arg-Thr-Arg-Tyr-Glu-Arg-Asn-Ile-Glu-Lys-Cys;
  • TRIP-12 SEQ ID NO:10: MPR-Cha-Val-Arg-Ile-Gly-Lys-
  • TRIP-13 SEQ ID NO:11: Leu-Val-Arg-I.le-Gly-Lys-His-
  • TRIP-15 SEQ ID NO:13: GBA-Ile-Gly-Lys-His-Ser-Arg- Thr-Arg-Tyr-Glu-Arg-Asn-Ile-Glu-Lys-Ile;
  • TRIP-16 Leu-Val-Arg-D-Cys-Gly-Lys-His-Ser-Arg-Thr- Arg-Tyr-Glu-Arg-Asn;
  • TRIP 17 SEQ ID NO:14: Arg-Ile-Gly-Lys-His-Ser-Arg- Thr-Arg-Tyr-Glu-Arg-Asn; TRIP 19: SEQ ID NO:15: Leu-Val-Arg-Ile-Gly-Lys-His- Ser-Arg;
  • TRIP 20 Leu-Val-Arg-D-Cys-Gly-Lys-His-Ser-Arg;
  • Alk-TRIP 20 Leu-Val-Arg-alkCys-Gly-Lys-His-Ser-Arg;
  • TRIP 21 Leu-Val-Arg-D-Ser-Gly-Lys-His-Ser-Arg
  • TRIP 22 SEQ ID NO:16: Leu-Val-Arg-Ser-Gly-Lys-His- Ser-Arg
  • TRIP 23 SEQ ID NO:17: Leu-Val-Arg-Met-Gly-Lys-His- Ser-Arg;
  • TRIP 24 Leu-Val-Arg-D-Cys-Gly-Lys-Tyr-Ala-Arg
  • TRIP 25 Leu-Val-Arg-D-Cys-Gly-Leu-His-Phe-Arg
  • TRIP 26 Val-Val-Arg-D-Cys-Gly-Lys-His-Phe-Arg;
  • TRIP 27 Leu-Val-Arg-D-Cys-Ser-Lys-His-Ser-Arg;
  • TRIP 28 Leu-Val-Arg-D-Cys-Thr-Lys-His-Ser-Arg;
  • TRIP 29 Leu-Val-Arg-D-Cys-Tyr-Lys-His-Ser-Arg
  • TRIP 30 Leu-Val-Arg-D-Cys-Asn-Tyr-His-Ser-Arg
  • TRIP 31 Leu-Val-Arg-D-Cys-Gln-Lys-His-Ser-Arg;
  • TRIP 32 Leu-Val-Arg-D-Cys-Asp-Lys-His-Ser-Arg;
  • TRIP 33 Leu-Val-Arg-D-Cys-Glu-Lys-His-Ser-Arg;
  • TRIP 34 Leu-Val-Arg-D-Cys-Lys-Lys-His-Ser-Arg
  • TRIP 35 Leu-Val-Arg-D-Cys-Arg-Lys-His-Ser-Arg
  • TRIP 36 Leu-Val-Arg-D-Cys-His-Lys-His-Ser-Arg
  • TRIP 37 Leu-Val-Arg-D-Cys-D-Ala-Lys-His-Ser-Arg
  • TRIP 38 Leu-Val-Arg-D-Cys-D-Ser-Lys-His-Ser-Arg;
  • TRIP 39 Leu-Val-Arg-D-Cys-D-Thr-Lys-His-Ser-Arg;
  • TRIP 40 Leu-Val-Arg-D-Cys-D-Tyr-Lys-His-Ser-Arg
  • TRIP 41 Leu-Val-Arg-D-Cys-D-Asn-Lys-His-Ser-Arg
  • TRIP 42 Leu-Val-Arg-D-Cys-D-Gln-Lys-His-Ser-Arg;
  • TRIP 43 Leu-Val-Arg-D-Cys-D-Asp-Lys-His-Ser-Arg;
  • TRIP 44 Leu-Val-Arg-D-Cys-D-Glu-Lys-His-Ser-Arg;
  • TRIP 45 Leu-Val-Arg-D-Cys-D-Lys-Lys-His-Ser-Arg
  • TRIP 46 Leu-Val-Arg-D-Cys-D-Arg-Lys-His-Ser-Arg
  • TRIP 47 Leu-Val-Arg-D-Cys-D-His-Lys-His-Ser-Arg;
  • TRIP 48 D-Cys-Arg-Leu-Ser-Val-His-Arg-Lys-Gly;
  • TRIP 49 SEQ ID NO:18: Leu-Val-Arg-Ile-Gly-Lys-His- Ser-Lys-Thr-Arg-Tyr-Glu-Lys-Asn; TRIP 50: SEQ ID NO:19: Leu-Val-Arg-Ile-Gly-Lys-His- Ser-His-Thr-Arg-Tyr-Glu-His-Asn;
  • TRIP 51 SEQ ID NO:20: Leu-Val-Arg-Ile-Gly-Lys-His- Ser-Ala-Thr-Arg-Tyr-Glu-Ala-Asn;
  • TRIP 52 SEQ ID NO:21: Leu-Val-Arg-Ile-Gly-Lys-His- Ser-Arg-Thr-Cys-Tyr-Glu-Arg-Asn;
  • TRIP-2, TRIP-4, TRIP-5, TRIP-11 and TRIP-12 each contains an intramolecular disulfide bond and are examples of cyclized thrombin receptor antagonists.
  • the disulfide bond is between the cysteine residues.
  • the disulfide bond is formed between the N-terminal mercaptopropionic acid, a non-amino acid component, and the C-terminal cysteine residue.
  • another embodiment of this invention is a fusion protein which comprises the formula: PP L -T-PP R ; wherein T is a thrombin receptor antagonist of this invention; and PP L and PP R are both portions of a polypeptide of the formula: PP L -BL-PP R ; wherein BL forms a ⁇ -loop structure in said polypeptide; and each of PP L and PP R are characterized by an antiparallel ⁇ -strand structure in said polypeptide.
  • a fusion protein initially involves identifying a naturally occurring protein which possesses a ⁇ -loop between two antiparallel ⁇ - strands structure.
  • Naturally occurring proteins are known to contain such a structure. These include erabutoxinA, both the heavy and light chain immunoglobulins; and serine proteases, such as trypsin. Any protein which has been crystallized can be scanned for such a loop/anti-parallel strand structure using X- ray crystallography.
  • the amino acids which make up the ⁇ -loop portion will be replaced with a peptide shown by the methods described above to act as a thrombin receptor antagonist.
  • This replacement will normally be done at the nucleotide level by recombinant DNA technology. Because of this, the gene or a cDNA encoding the naturally occurring protein must be available. Also, the ⁇ -loop replacement i.e., the thrombin receptor antagonist peptide, must be made up entirely of naturally occurring amino acids and a synthetic gene encoding that antagonist must be synthesized.
  • a fusion protein The manipulations required to construct such a fusion protein are commonly employed in the art. Typically, this involves removing the naturally occurring ⁇ -loop coding region via restriction endonuclease digestions, exonuclease digestions or a combination of both. The remainder of the DNA encoding that protein is then isolated and ligated to a synthetic gene encoding the antagonist. Large quantities of the DNA encoding the desired fusion protein may be obtained via PCR (polymerase chain reaction) or by conventional cloning techniques. Alternately, the gene encoding the desired fusion protein may be constructed synthetically by piecing together various oligonucleotides.
  • the fusion protein is subsequently produced by transformation or transfection of an appropriate host with the above DNA.
  • Those of skill in the art are well aware of the factors necessary to achieve sufficient expression of the fusion protein. These include the operative linkage of the DNA encoding the fusion protein to an appropriate promoter, choice of appropriate host, proper growth conditions, etc.
  • the antiplatelet activity of the antagonists of this invention may be measured by any of a number of conventional, in vitro platelet assays.
  • the assay will measure a change in the degree of aggregation of platelets or a change in the release of a platelet secretory component in the presence of thrombin.
  • the former may be measured in an aggregometer.
  • the latter may be measured using RIA or ELISA techniques specific for the secreted component.
  • the inability of the antagonists of this invention to inhibit thrombin's catalytic activity may be assayed in vitro utilizing a synthetic peptidyl substrate with a colorimetric marker.
  • a synthetic peptidyl substrate with a colorimetric marker.
  • An example of such a substrate is Spectrozyme TH (tosyl-Gly-Pro-Arg- p-nitroanilide; American Diagnostica, New York, NY) . Cleavage of that substrate can be quantitated using a spectrophotometer. Inhibition of thrombin's catalytic activity may also be assayed using an in vitro assay of fibrinogen cleavage.
  • an antagonist of this invention to bind to the anionic domain of the thrombin receptor is easily determined in a competition assay. For example, incubation of thrombin receptor-containing cells (platelets, smooth muscle cells, endothelial cells, etc.) with a radiolabelled antagonist in the presence or absence of an unlabelled peptide corresponding to the thrombin receptor anionic domain ("thrombin receptor peptide") provides a quantification of such binding. Alternatively,. affinity chromatography of the labelled antagonist on a column containing a thrombin receptor peptide cross-linked to an insoluble matrix could be used to assay binding.
  • thrombin receptor antagonists of the present invention may be synthesized by various techniques which are well known in the art. These include enzymatic cleavage of thrombin, recombinant DNA techniques, solid-phase peptide synthesis, solution- phase peptide synthesis, organic chemical synthesis techniques, or a combination of these techniques. The choice of synthesis technique will, of course, depend upon the composition of the particular antagonist.
  • the thrombin antagonist is entirely peptidic and is synthesized by solid-phase peptide synthesis techniques. solution-phase peptide synthesis techniques or a combination thereof which constitute the most cost- efficient procedures for producing commercial quantities of these antagonists.
  • non-protein amino acids When “non-protein” amino acids are contained in the thrombin receptor antagonist, they may be either added directly to the growing chain during peptide synthesis or prepared by chemical modification of the complete synthesized peptide, depending on the nature of the desired "non-protein” amino acid. Those of skill in the chemical synthesis art are well aware of which "non-protein” amino acids may be added directly and which must be synthesized by chemically modifying the complete peptide chain following peptide synthesis.
  • thrombin antagonists of this invention which contain both non-amino acid and peptidic portions is preferably achieved by a mixed heterologous/solid phase technique.
  • This technique involves the solid-phase synthesis of all or most of the peptide portion of the molecule. This is followed by the addition of the non-amino acid components which are synthesized by solution phase techniques and then coupled to the peptidic portion via solid-phase or solution-phase methods. Any remaining peptidic portions may then be added via solid-phase or solution- phase methods.
  • the thrombin receptor antagonists of the present invention are useful in compositions and methods for the treatment and prophylaxis of various diseases attributed to thrombin-mediated and thrombin- associated functions and processes. These include myocardial infarction, stroke, pulmonary embolism, peripheral arterial occlusion, restenosis following arterial injury or invasive cardiological procedures, acute or chronic atherosclerosis, edema and inflammation, various cell regulatory processes (e.g. secretion, shape changes, proliferation) , and neurodegenerative diseases.
  • diseases attributed to thrombin-mediated and thrombin- associated functions and processes include myocardial infarction, stroke, pulmonary embolism, peripheral arterial occlusion, restenosis following arterial injury or invasive cardiological procedures, acute or chronic atherosclerosis, edema and inflammation, various cell regulatory processes (e.g. secretion, shape changes, proliferation) , and neurodegenerative diseases.
  • the thrombin receptor antagonists of the present invention may be formulated using conventional methods to prepare pharmaceutically useful compositions, such as the addition of a pharmaceutically acceptable carrier. These compositions and the methods employing them may be used for treating or preventing platelet activation in a patient. Inhibition of thrombin-induced platelet activation is particularly useful in the treatment and prophylaxis of platelet-dependent (arterial) thrombosis.
  • compositions of this invention may be administered to the patient in any pharmaceutically acceptable dosage form, including those which may be administered to a patient intravenously as bolus or by continued infusion, intramuscularly — including paravertebrally and periarticularly — subcutaneously, intracutaneously, intra-articularly, intrasynovially, intrathecally, intra-lesionally, periostally or by oral, nasal, or topical routes.
  • Such compositions are preferably adapted for topical, nasal, oral and parenteral administration, but, most preferably, are formulated for parenteral administration.
  • Parenteral compositions are most preferably administered as a constant infusion.
  • fluid unit dose forms are prepared which contain a thrombin receptor antagonist of the present invention and a sterile vehicle.
  • the antagonist may be either suspended or dissolved, depending on the nature of the vehicle and the nature of the particular antagonist.
  • Parenteral compositions are normally prepared by dissolving the thrombin receptor antagonist in a vehicle, optionally together with other components, and filter sterilizing before filling into a suitable vial or ampule and sealing.
  • adjuvants such as a local anesthetic, preservatives and buffering agents are also dissolved in the vehicle.
  • the composition may then be frozen and lyophilized to enhance stability.
  • Parenteral suspensions are prepared in substantially the same manner, except that the active component is suspended rather than dissolved in the vehicle. Sterilization of the compositions is preferably achieved by exposure to ethylene oxide before suspension in the sterile vehicle.
  • a surfactant or wetting agent is included in the composition to facilitate uniform distribution of its components.
  • Tablets and capsules for oral administration may contain conventional excipients, such as binding agents, fillers, diluents, tableting agents, lubri ⁇ cants, disintegrants, and wetting agents.
  • the tablet may be coated according to methods well known in the art.
  • Suitable fillers which may be employed include cellulose, mannitol, lactose and other similar agents.
  • Suitable disintegrants include, but are not limited to, starch, polyvinylpyrrolidone and starch derivatives, such as sodium starch glycolate.
  • Suitable lubricants include, for example, magnesium stearate.
  • Suitable wetting agents include sodium lauryl sulfate.
  • Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or another suitable vehicle before use.
  • Such liquid preparations may contain conventional additives.
  • suspending agents such as sorbitol, syrup, methyl cellulose, gelatin, hydroxyethyl-cellulose, carboxy- methylcellulose, aluminum stearate gel or hydrogenated edible fats; emulsifying agents which include lecithin, sorbitan monooleate, polyethylene glycols, or acacia; non-aqueous vehicles, such as almond oil, fractionated coconut oil, and oily esters; and preservatives, such as methyl or propyl p-hydroxybenzoate or sorbic acid.
  • Compositions formulated for topical administration may, for example, be in aqueous jelly, oily suspension or emulsified ointment form.
  • the dosage and dose rate of the thrombin receptor antagonist will depend on a variety of factors, such as the size of the patient, the specific pharmaceutical composition used, the object of the treatment, i.e., therapy or prophylaxis, the nature of the condition to be treated, and the judgment of the treating physician.
  • a pharmaceutically effective dosage is defined herein as being between about 0.001 and 100 mg/kg body weight of the patient to be treated. It should, however, be understood that other dosages may be useful based on the factors enumerated above.
  • a maintenance dose of a composition of this invention is administered, if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment upon any recurrence of disease symptoms.
  • the thrombin receptor antagonist may be employed in compositions and methods for inhibiting thrombin-induced smooth muscle cell proliferation in a patient.
  • the thrombin-induced multiplication of intimal smooth muscle cells which line the outer surface of blood vessels has been implicated in advanced atherosclerosis and restenosis following angioplasty [D. T. Hung et al., J. Cell Biol.. 116, pp. 827-32 (1992); A. J. B. Brady et al., Brit. J. Med.. 303, pp. 729-30 (1991)].
  • the inhibition of smooth muscle cell proliferation in a patient is achieved by inserting into said patient a device through which is conveyed a composition comprising the thrombin receptor antagonist of this invention.
  • a portion of the device (preferably the end) must contain aperture means which will allow the antagonist to exit the device.
  • Such devices are well known in the art and are exemplified by balloon catheters.
  • a preferred embodiment of this method employs a weeping balloon catheter as the device which contains the thrombin receptor antagonist composition
  • thrombin receptor antagonist composition see for example, United States patent 5,049,132 and Wolinsky et al., "Use of a Perforated Balloon Catheter to Deliver Concentrated Heparin Into the Wall of the Normal Canine Artery", J. Amer. Coll. Cardiol.. 15, pp. 475-81 (1990) , the disclosures of which are hereby incorporated by reference] .
  • thrombin receptor antagonists may be utilized in methods for treating or preventing bone resorption. Osteoclasts are the cells responsible for bone resorption in the process of turnover and remodelling of bone.
  • thrombin is known to be stimulated by thrombin through a receptor on the osteoclast cell surface [D. N. Tatakis et al. , Biochem. Biophvs. Res. Comm.. 174, pp. 181-88 (1991)].
  • thrombin-induced osteoclast activity By inhibiting thrombin-induced osteoclast activity, bone resorption is decreased. This may be specifically useful in the treatment of osteoporosis and other conditions characterized by abnormally high levels of bone resorption and demineralization.
  • This invention also relates to methods which employ the above-described thrombin receptor antagonists to inhibit thrombin-induced inflammation.
  • Thrombin is known to induce the synthesis of platelet activation factor (PAF) by endothelial cells [S. Prescott et al.
  • PAF platelet activation factor
  • thrombin exerts this effect by binding to its receptor on the endothelial cell surface.
  • diseases include, but are not limited to, adult respiratory distress syndrome, septic shock, septicemia and reperfusion damage.
  • the thrombin receptor antagonists of this invention may also be employed in methods for treating neurodegenerative diseases.
  • Thrombin is known to cause neurite retraction, a process suggestive of the rounding in shape changes of brain cells and implicated in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. It is believed that thrombin acts on neuroblasts through a specific receptor on the surface of those cells [D. G ⁇ rwitz et al., Proc. Natl. Acad. Sci. USA. 85, pp. 3440-44 (1988)].
  • the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.
  • the resins employed to synthesize the various TRIPs were dependent upon the C-terminal amino acid of the antagonist. Specifically, the resins used were t-BOC- L-Glu(OBzl) (OCH 2 )-PAM, t-BOC-D-Cys(4-MeBzl) (-OCH 2 )-PAM, t-BOC-L-Ile(OCH 2 )-PAM, t-BOC-Gly(0CH 2 )-PAM, t-BOC-L- Cys(4-MeBzl) (-OCH 2 )-PAM, t-BOC-L-Arg(Mts) and t-BOC-L- Asn.
  • the resins used were t-BOC- L-Glu(OBzl) (OCH 2 )-PAM, t-BOC-D-Cys(4-MeBzl) (-OCH 2 )-PAM, t-BOC-L-
  • Resins were purchased from either Applied Biosystems or Peninsula Laboratories (Belmont, CA) . We employed various protected amino acids for peptide synthesis depending upon the sequence of amino acids in the particular TRIP.
  • the protected amino acids used in the syntheses were t-BOC-L-Leu, t-BOC-L- Val, t-BOC-Arg(Mts) , t-BOC-L-Ile(l/2 H 2 0) , t-BOC-Gly, t-BOC-L-Lys(2-Cl-z) , t-BOC-L-Ser(Bzl) , t-BOC-L-
  • TRIP-1 (SEQ ID NO:2: Leu-Val-Arg-Ile-Gly-Lys- His-Ser-Arg-Thr-Arg-Tyr-Glu-Arg-Asn-Ile-Glu) was synthesized using t-BOC-L-Glu(OBzl) (OCH 2 )-PAM resin and the appropriate protected amino acids selected from those listed in Example 1. After completion of synthesis, the peptide was fully deprotected and uncoupled from the resin by treatment with anhydrous HF: p_-cresol: ethylmethyl sulfate (10:1:1, v/v/v) . Peptides were then extracted from the resin with 5% acetic acid and lyophilized to dryness.
  • Crude TRIP-1 was purified by reverse-phase HPLC employing an Applied Biosystems 15IA liquid chromatographic system and a Vydac C18 column (2.2 x 25 cm). The column was equilibrated in 0.1% trifluoroacetic acid (TFA) in water and developed with a linear gradient of increasing acetonitrile concentration (0 to 80%) in 0.1% TFA over 45 minutes at a flow rate of 4.0 ml/min. The effluent stream was monitored at 229 nm for absorbance. Fractions were collected manually. Fractions containing TRIP-1 were pooled and lyophilized to dryness. Typically, 20-50 mg of crude TRIP-1 was loaded onto the column and yielded 5-35 mg of purified peptide.
  • TFA trifluoroacetic acid
  • TRIP-1 All of the subsequent TRIPs described below were synthesized in the same manner as that described for TRIP-1, using the appropriate resin and protected amino acids, unless otherwise indicated. Similarly, all of the below-described TRIPs were uncoupled, deprotected, purified and analyzed in the same manner as that described for TRIP-1.
  • TRIP-2 (Leu-Val-Arg-Ile-Cys-Lys-His-Ser-Arg- Thr-Arg-Tyr-Glu-Arg-Asn-Ile-(D-Cys) ) was synthesized in the same manner as TRIP-1, using the appropriate resin and protected amino acids. Uncoupling, deprotection, purification and analysis were also performed as described for TRIP-1.
  • Cyclized TRIP-2 was purified by the same reverse-phase HPLC chromatography as described for the purification of TRIP-1. The structure of the cyclic TRIP-2 was confirmed by FAB-mass spectroscopy. The parent ion molecular mass for cyclized TRIP-2 was 2 units lower that the mass for the linear TRIP-2. This indicated that the oxidation of thiols to yield a disulfide had occurred.
  • TRIP-3 TRIP-3 has the formula: SEQ ID NO:3: Leu-
  • TRIP-4 has the formula: Leu-Val-Arg-(D-Cys)-
  • TRIP-5 has the formula: Leu-Val-Arg-(D-Cys) Gly-Lys-His-Ser-Arg-Thr-Arg-Tyr-Glu-Arg-Asn-(D-Cys)- Glu-Lys-Ile. TRIP-5 was synthesized and was cyclized as described above.
  • TRIP-6 has the formula: SEQ ID NO:4: Leu- Val-Arg-Ile-Gly-Lys-His-Ser-Arg-Thr-Arg-Tyr-Glu-Arg- Asn-Ile-Glu-Lys-Ile and was synthesized as described above.
  • TRIP-7 has the formula: SEQ ID NO:5: Leu-
  • Val-Arg-Ile-Gly-Orn-His-Ser-Arg-Thr-Arg-Tyr-Glu-Arg- Asn-Ile-Glu-Lys-Ile was synthesized as described above.
  • TRIP-8 has the formula: SEQ ID NO:6: Leu- Val-Arg-Ile-Gly-Lys-His-Ser-Arg-Thr-Arg-Tyr-Asp-Arg- Asn-Ile-Glu-Lys-Ile and is synthesized as described above.
  • TRIP-9 has the formula: SEQ ID NO:7: Leu- Val-HArg-Ile-Gly-Lys-His-Ser-Arg-Thr-Arg-Tyr-Glu-Arg- Asn-Ile-Glu-Lys-Ile and was synthesized as described above. Boc-HArg was synthesized by conventional procedures well known in the protein synthesis art.
  • TRIP-10 has the formula: SEQ ID NO:8: Leu- Val-Arg-Ile-Gly-Lys-His-Ser-Arg-Thr-Arg-Tyr-Glu-Arg- Asn-Ile-Glu-Lys-Cha-Gly and was synthesized as described above.
  • TRIP-11 has the formula: SEQ ID NO:9: MPR-
  • Residues 2-19 of this antagonist are synthesized by conventional peptide synthesis procedures as described in Example 2.
  • the peptide resin was then reacted with a 100-fold molar excess of mercaptopropionic acid (MPR) using the conventional coupling procedure of the Applied Biosystems peptide synthesizer.
  • MPR mercaptopropionic acid
  • the successful coupling of MPR to the antagonist was tested by ninhydrin analysis of an aliquot of the reacted resin. Absence of a blue color in the ninhydrin reaction indicates successful incorporation of MPR. If a positive reaction occurs (blue color) , an additional cycle of MPR addition was performed. Cyclization of TRIP-11 was performed as described in Example 3, following synthesis of the complete molecule.
  • TRIP-12 TRIP-12 has the formula: SEQ ID NO:10: MPR-
  • TRIP-12 was synthesized in the same manner described above for TRIP-11, except that residues 2-20 are synthesized by conventional solid phase synthesis prior to MPR addition. TRIP-12 was cyclized as described above.
  • TRIP-13 has the formula: SEQ ID NO:11: Leu- Val-Arg-Ile-Gly-Lys-His-Ser-Arg-Cha-Arg-Tyr-Glu-Arg- Asn-Ile-Glu-Lys-Ile and was synthesized as described above.
  • TRIP-14 has the formula: SEQ ID NO:12: Leu- Val-Arg-Ile-Gly-Lys-His-Ser-Arg-Thr-Arg-Tyr(I 2 )-Glu- Arg-Asn-Ile-Glu-Lys-Ile and was synthesized as described above.
  • TRIP-15 has the formula: SEQ ID NO:13: GBA- Ile-Gly-Lys-His-Ser-Arg-Thr-Arg-Tyr-Glu-Arg-Asn-Ile- Glu-Lys-Ile.
  • Residues 2-16 of TRIP-15 are synthesized by the conventional solid phase techniques described in Example 2.
  • the peptide resin was then reacted with a 100-fold molar excess of para-guanidinobenzoic acid (GBA) using the conventional coupling procedure of the Applied Biosystems peptide synthesizer.
  • GBA para-guanidinobenzoic acid
  • Successful addition of GBA was confirmed by the ninhydrin test described for TRIP-11 (Example 12) .
  • Platelet Aggregation Assays Platelet-rich plasma (PRP) was obtained from healthy human volunteers who had refrained from taking aspirin for at least one week prior to the taking of samples. Blood was collected into 1/10 volume of 3.8% trisodium citrate using a 21 gauge butterfly cannula. The blood was then centrifuged at room temperature for 15 minutes at 100 x g and the supernatant was collected. The supernatant contained 250,000 - 400,000 platelets/ ⁇ l.
  • Platelet aggregation studies were performed with gel-filtered platelets using a 4-channel platelet aggregation profiler (PAP4, Biodata, Hatboro, PA). Specifically, the assay was performed as follows: Varying concentrations of TRIP-1, (0 - 1 mM final) were added to 0.45 ml of a gel-filtered platelet suspension. The mixture was then placed in the platelet aggregometer to initiate warming to 37°C and stirring. After equilibration (approximately 5 minutes) we added human thrombin (0.16-0.48 U/ml final) to the mixture. The aggregation response was monitored for 5 minutes. Inhibition was calculated by the following formula:
  • Figure 1 demonstrates that TRIP-1 inhibited thrombin-induced platelet aggregation in a dose- dependent manner at varying thrombin.concentrations. At a concentration of 800 ⁇ M, TRIP-1 inhibited thrombin-induced platelet aggregation by >80% for all concentrations of thrombin tested.
  • thrombin receptor antagonists of this invention were also analyzed the ability of the thrombin receptor antagonists of this invention to inhibit platelet aggregation induced by the tethered ligand (5 ⁇ M) , collagen (76 ⁇ g/ml) , or ADP (4 ⁇ M) .
  • the gel filtered platelet suspension was supplemented with purified human fibrinogen (1 mg/ml) .
  • concentrations of antagonist that fully inhibited thrombin-induced aggregation 800 ⁇ M for TRIP-1) . The results of this experiment are depicted in Figures 2 and 3.
  • FIG. 2 demonstrates that TRIP-1, at a concentration which completely inhibited thrombin- induced platelet aggregation, had no inhibitory effect on platelet aggregation induced by the tethered ligand peptide.
  • Figure 3 shows that TRIP-1 was specific for inhibiting thrombin-induced platelet aggregation and had no effect on platelets induced by ADP or collagen.
  • an antagonist of this invention to bind to the hirudin-like anionic domain of the thrombin receptor was assayed by first labelling the antagonist with ,25 I. The labelled antagonist was then incubated with cells containing the thrombin receptor, such as platelets or smooth muscle cells. The thrombin receptor-containing cells may be maintained in either a suspension culture or grown adherent on plastic or another suitable substrate.
  • Binding of the antagonist to the anionic domain of the thrombin receptor was demonstrated by (1) specific binding of the labelled antagonist to the cells; and (2) significantly decreased binding of the labelled antagonist to the same cells when incubated in the presence of an unlabelled peptide corresponding to the anionic domain of the thrombin receptor (amino acids 49-62; "thrombin receptor peptide") .
  • the thrombin receptor peptide may be cross-linked to an insoluble support, such as CNBr-activated Sepharose. This complex may then serve as an affinity matrix in a column for the binding of an I25 I-labelled antagonist.
  • an antagonist of this invention to such a matrix (and therefore to the anionic domain of the thrombin receptor) was confirmed by adsorbance to the column. The antagonist will then be specifically eluted from the column with soluble, unlabelled thrombin receptor peptide.
  • TRIP-6 10 amino acids of TRIP-6 could be replaced with either glycine or alanine (or, at some position with both) without decreasing inhibition to less than 50% of that observed for TRIP-6. This suggested that these amino acids could be deleted from the molecule without destroying inhibitory properties. Moreover, smaller peptides are more desirable for therapeutic uses due to decreased antigenicity.
  • TRIP-16 has the formula: Leu-Val-Arg-D-Cys- Gly-Lys-His-Ser-Arg-Thr-Arg-Tyr-Glu-Arg-Asn and was synthesized as described above.
  • TRIP 17 has the formula: SEQ ID NO:14: Arg- Ile-Gly-Lys-His-Ser-Arg-Thr-Arg-Tyr-Glu-Arg-Asn and was synthesized as described above.
  • EXAMPLE 23 Synthesis Of TRIP-19 TRIP 19 has the formula: SEQ ID NO:15: Leu- Val-Arg-Ile-Gly-Lys-His-Ser-Arg and was synthesized as described above.
  • TRIP-20 has the formula: Leu-Val-Arg-D-Cys- Gly-Lys-His-Ser-Arg and was synthesized as described above.
  • Alk-TRIP 20 has the formula: Leu-Val-Arg- alkCys-Gly-Lys-His-Ser-Arg and was synthesized as described in Example 24. We then incubated 1 mM of the peptide with 1.1 mM iodoacetamide for 30 minutes at room temperature. The alkylated product was isolated by reverse-phase C-18 chromatography. The identity of the product was confirmed by mass spectroscopy as described above and by failure to produce a colored product with Ellman's reagent (G. E. Means and R. E. Feeney, "Chemical Modification of Protein", Holden-Day, Inc., San Francisco, CA, pp. 107, 155).
  • TRIP 21 has the formula: Leu-Val-Arg-D-Ser- Gly-Lys-His-Ser-Arg and was synthesized as described above.
  • TRIP 22 has the formula: SEQ ID NO:16: Leu- Val-Arg-Ser-Gly-Lys-His-Ser-Arg and was synthesized as described above.
  • TRIP 23 has the formula: SEQ ID NO:17: Leu- Val-Arg-Met-Gly-Lys-His-Ser-Arg and was synthesized as described above.
  • TRIP-24 has the formula: Leu-Val-Arg-D-Cys- Gly-Lys-Tyr-Ala-Arg and is synthesized as described above.
  • TRIP-25 has the formula: Leu-Val-Arg-D-Cys- Gly-Leu-His-Phe-Arg and is synthesized as described above.
  • TRIP-26 has the formula: Val-Val-Arg-D-Cys- Gly-Lys-His-Phe-Arg and is synthesized as described above.
  • TRIP 27 has the formula: Leu-Val-Arg-D-Cys- Ser-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP-28 has the formula: Leu-Val-Arg-D-Cys- Thr-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP 29 has the formula: Leu-Val-Arg-D-Cys- Tyr-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP 30 has the formula: Leu-Val-Arg-D-Cys- Asn-Tyr-His-Ser-Arg and is synthesized as described above.
  • TRIP 31 has the formula: Leu-Val-Arg-D-Cys- Gln-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP-32 has the formula: Leu-Val-Arg-D-Cys- Asp-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP 33 has the formula: Leu-Val-Arg-D-Cys- Glu-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP-34 has the formula: Leu-Val-Arg-D-Cys- Lys-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP-35 has the formula: Leu-Val-Arg-D-Cys- Arg-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP-36 has the formula: Leu-Val- ⁇ rg-D-Cys- His-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP-37 has the formula: Leu-Val-Arg-D-Cys-D- Ala-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP 38 has the formula: Leu-Val-Arg-D-Cys-D- Ser-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP 39 has the formula: Leu-Val-Arg-D-Cys-D- Thr-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP-40 TRIP 40 has the formula: Leu-Val-Arg-D-Cys-D- Tyr-Lys-His-Ser-Arg and is synthesized as described above.
  • EXAMPLE 46 Synthesis Of TRIP-41 TRIP 41 has the formula: Leu-Val-Arg-D-Cys-D- Asn-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP-42 has the formula: Leu-Val-Arg-D-Cys-D- Gln-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP 43 has the formula: Leu-Val-Arg-D-Cys-D- Asp-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP 44 has the formula: Leu-Val-Arg-D-Cys-D- Glu-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP 45 has the formula: Leu-Val-Arg-D-Cys-D- Lys-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP-46 has the formula: Leu-Val-Arg-D-Cys-D- Arg-Lys-His-Ser-Arg and is synthesized as described above.
  • EXAMPLE 52 Synthesis Of TRIP-47 TRIP 47 has the formula: Leu-Val-Arg-D-Cys-D- His-Lys-His-Ser-Arg and is synthesized as described above.
  • TRIP 48 has the formula: D-Cys-Arg-Leu-Ser- Val-His-Arg-Lys-Gly and is synthesized as described above.
  • TRIP 49 has the formula: SEQ ID NO:18: Leu- Val-Arg-Ile-Gly-Lys-His-Ser-Lys-Thr-Arg-Tyr-Glu-Lys-Asn and is synthesized as described above.
  • TRIP-50 TRIP 50 has the formula: SEQ ID NO:19: Leu- Val-Arg-Ile-Gly-Lys-His-Ser-His-Thr-Arg-Tyr-Glu-His-Asn and is synthesized as described above.
  • TRIP 51 has the formula: SEQ ID NO:20: Leu- Val-Arg-Ile-Gly-Lys-His-Ser-Ala-Thr-Arg-Tyr-Glu-Ala-Asn and is synthesized as described above.
  • TRIP-52 has the formula: SEQ ID NO:21: Leu- Val-Arg-Ile-Gly-Lys-His-Ser-Arg-Thr-Cys-Tyr-Glu-Arg-Asn and is synthesized as described above.
  • EXAMPLE 58 Platelet Aggregation Assay Of Other TRIPs We assayed the ability of TRIP-2 through TRIP-21 (excluding TRIP-9) to inhibit thrombin-induced platelet aggregation in the assay described in Example 17. The results of these assays are set forth in the table below.
  • TRIP-19 replacement of the lie residue in TRIP-19 with a D-Cys (TRIP-20) produces a greater than 20-fold increase in inhibitory activity and results in a molecule that is also 6-fold more potent than the longer TRIP-6. This confirms the relative unimportance of thrombin amino acids 74-83 in binding to the thrombin receptor.
  • thrombin receptor antagonists of this invention also fail to inhibit thrombin's catalytic activity.
  • TRIPs Do Not Inhibit Thrombin Polymerization of Fibrin
  • the ability of the thrombin receptors of this invention to inhibit thrombin's cleavage of fibrinogen to form fibrin was measured indirectly in a turbidimetric assay for fibrin polymerization.
  • Thrombin (0.11 U/ml) was added to a human fibrinogen solution (1 mg/ml) at 25°C in 50 mM Tris, 100 mM NaCl, pH 8 containing buffer (control) , TRIP-6 (100 ⁇ M) , or HIRULOGTM (9 nM) . Onset of change in turbidity ("onset time") was recorded in a 96-well plate reader
  • inhibition of thrombin-induced cellular functions is assayed by pre-incubating target cells with varying concentrations of a thrombin receptor antagonist of this invention (0 - 1 mM) for 5- 30 minutes. We then add thrombin to these cells for 5- 30 minutes to stimulate a given cellular response. The cells and/or media are then collected and assayed for a known thrombin-induced component. Samples incubated with TRIP-1 are compared to control samples (no inhibitor) to quantify inhibition.
  • Thrombin is known to induce increased neutrophil adhesiveness in endothelial cells.
  • PAF platelet-activating factor
  • Polymorphonuclear leukocytes are prepared by centrifuging citrated whole blood (see Example 17) through a commercially available ficoll gradient (Pharmacia, Piscataway, NJ) . Specifically, 30 ml of whole blood is slowly pipetted down the side of the tube containing the gradient, being careful not to disturb the interface. The gradient is centrifuged for 40 minutes at 1600 rpm at 22°C. The pellet containing the PMNs is resuspended in 30 ml of white blood cell media (prepared fresh weekly) containing 25 mM HEPES, pH 7.3, with 0.5% bovine serum albumin in HBSS (without calcium and magnesium) .
  • the PMNs are then labelled for 30 minutes at 37°C by the addition of 3.0 ml of BCECF-AM (2' ,7'-bis-(2-carboxyethyl)-5(and - 6)-carboxyfluorescein acetoxymethyl ester) (Molecular Probes, Inc., Eugene, OR).
  • BCECF-AM (2' ,7'-bis-(2-carboxyethyl)-5(and - 6)-carboxyfluorescein acetoxymethyl ester
  • the cells are then diluted by the addition of 5 volumes of white blood cell media.
  • Human umbilical vein endothelial cells are grown in 48-well plates for 3 days until reaching confluency.
  • the HUVECs are then washed with HBSS, followed by the addition of 0 - 1 mM thrombin receptor antagonist.
  • the cells are then incubated at 37°C for 10-30 minutes.
  • the PMN suspension (0.5 ml) is equilibrated to 37°C, added to HUVEC culture and allowed to settle for 2 minutes.
  • Thrombin (0 - 1 U/ml) is then added to the cell mixture which is allowed to incubate for an additional 10 minutes at 37°C.
  • the wells are washed several times with HBSS to remove non-adherent PMNs.
  • the wells are next treated with 0.3 ml of 1% NP- 40 and the contents of each well are transferred to a separate 96-well plate. Fluorescence measurements are carried out on the NP-40-treated-material using an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Fluorescence is proportional to the number of PMNs that adhere to the HUVEC monolayer. Pre-incubation with thrombin receptor antagonist reduces the mean fluorescence as compared to control wells containing thrombin, but no inhibitor.
  • GMP-140 PAF generation and cell surface expression of GMP-140 are assayed as described previously [S. Prescott et al. , Proc. Natl. Acad. Sci. USA. 81, pp. 3534-38 (1984)].
  • rat vascular smooth muscle cells A10, rat smooth muscle embryonic thoracic aorta; ATCC 1476-CRL. Batch #F9432
  • DMEM fetal calf serum
  • thrombin receptor antagonists of this invention exhibit a dose-dependent inhibition of 3 H-thymidine incorporation into smooth muscle cells. This confirms that these thrombin receptor antagonists inhibit the thrombin receptor on the smooth muscle cell surface.
  • the erabutoxinA-TRIP fusion protein was designed to contain the following structural features, listed in N- to C-terminal order: the signal peptide from phage M13 PHI protein; amino acids 1-24 of erabutoxin-A; a sequence corresponding to amino acids 3-18 of TRIP-6 (amino acids 3-18 of SEQ ID N0:4); erabutoxin-A amino acids 30-57, a linker of two alanine residues; a sequence recognized by the anti-Cmyc antibody 9E10; a sequence encoding a Factor Xa cleavage site; and the full length sequence of phage M13 PHI protein, starting at the first residue of the mature molecule.
  • the ErabutoxinA/TRIP fusion gene was constructed as a synthetic gene from oligonucleotide fragments shown below. All oligonucleotides were synthesized using an Applied Biosystems Model 394 oligonucleotide synthesizer (Applied Biosystems, Foster City, CA) .
  • PHI signal sequence (nucleotides 2665-2733), the first 24 amino acids of erabutoxinA (nucleotides 2734-2820), the TRIP sequence (nucleotides 2821-2868) , amino acids 30-57 of erabutoxinA (nucleotides 2869-2952) , an Ala-Ala linker (nucleotides 2953-2958) , a region coding for an epitope recognized by the anti-Cmyc antibody 9E10 (nucleotides 2959-2987) , a region coding for a Factor Xa cleavage site (nucleotides 2988-3015) and the PHI protein coding sequence (nucleotides 3016-4233) .
  • This vector also contained a HindHI site (nucleotide 2657) and a Notl site (nucleotide 2955) .
  • the sequence of the predicted protein encoded by this construct begins with the PHI signal sequence Leu-Glu-Ile-Phe-Asn and ends with Arg-Asn-Lys-Glu-Ser.
  • the signal peptide sequence was presumed to be removed, creating a new N-terminus which was residue 1 of erabutoxin-A.
  • amino acids 25-29 (Ser-Asp-Phe-Arg-Gly) of erabutoxin-A is replaced by the TRIP sequence.
  • Competent DH5ocF'lq E. coli cells were transformed with the ligated pTZ19R/Erabutoxin-A-
  • TRIP/PIII construct and plated on ampicillin (50 ⁇ g/ml) LB plates. After colonies appeared, a single colony was picked and used to inoculate 1.5 ml of 2xYT media containing 100 ⁇ g AMP and 2% glucose. The transformant were grown with vigorous shaking for 4 to 6 hours at 37°C until reaching an OD ⁇ o of about 0.9. The cells were then centrifuged and the cell pellet was resuspended in 6 ml 2xYT together with 1 x 10" colony forming units (CFU) of phage M13K07 and incubated on ice for 5-10 min.
  • CFU colony forming units
  • the infected cells were transferee to a 250 ml flask and grown with vigorous shaking at 30°C for 1 hr. Phage production was stimulated by adding IPTG (10 ⁇ M) , ampicillin (80 ⁇ g/ml) and kanamycin (83 ⁇ g/ml) . Cultures were grown for 14-18 hours at 37°C. We then centrifuged the cells and titered the supernatant for phage. Phage prepared in this manner may be stored at 4°C.
  • the 5' segment of this modified construct was made by amplifying pTZ19R/erabutoxinA-TRIP with oligonucleotides ST11 and SRP24. Three different 3' segments were made using oligonucleotide SRPIOA and one of oligonucleotides ST8, ST9 or ST10 to make each of three variants in the TRIP sequence. Following PCR, the 5' and 3' segments were isolated on an agarose gel, and recovered using the Gene Clean Kit (Bio 101, San Diego, CA) . These overlapping segments were then joined by the splice overlap extension method using the oligonucleotides SRP24 and SRPIOA. The resulting fragments were isolated as described above, then digested with Hindlll and Notl and cloned back into the parent vector. The sequences of the above-described oligonucleotides are:
  • SRPIOA [SEQ ID NO:36] CTT TCA ACA GTT TCT GCG GCC CCA
  • the resulting fusion proteins contained modified TRIP sequences characterized by an Arg-to-Lys, and Arg-to-His or an Arg-to-Ala double substitution at amino acids 9 and 14 of TRIP-6 [SEQ ID NO:4].
  • a mutational event also produced an additional Arg-to-Cys substitution at amino acid 11 of the Arg-to-Lys double substituted TRIP sequence.
  • Phage expressing each of the ErabutoxinA/TRIP fusion proteins described in Example 65 on their surface were prepared as described in Example 64 and then concentrated by adding 1/5 volume of 20% PEG 8000, incubating overnight at 4°C, then centrifuging at 15,000xg for 15 minutes.
  • the phage pellet was resuspended in 100 ⁇ l of Tris buffered saline (TBS, 100 mM TRIS, 150 mM NaCl) . Titers of the concentrated phage ranged between 2 - 9 x 10 12 CFU/ml.
  • a peptide, Asn-Asp-Lys-Tyr-Glu-Pro-Phe-Trp-Glu-Asp-Glu-Glu-Lys- Asn-Glu-Ser-Gly-Gly-Gly-Cys [SEQ ID NO:37], corresponding to the hirudin like region of the thrombin receptor was coupled to bovine serum albumin ("BSA") at a ratio of 2 mg peptide:5 mg BSA by first reacting the 5 mg of BSA with 0.25 mg SPDP (Pierce Chemical Company, Rockford II) for 1 hr at room temperature then adding the peptide and incubating an additional hour.
  • BSA bovine serum albumin
  • Plastic microtiter plates were coated with 2 ⁇ g/ml of thrombin receptor peptide-BSA conjugate in 0.1 M NaH 2 C0 3 pH 9.6 for 1 hr, then blocked with 5% BSA in TBS. After washing the plate three times with 0.1% TWEEN-20 in TBS (TBS-Tween) , varied concentrations of phage diluted in TBS-Tween were added. After 1 hour, the plate was washed and polyclonal rabbit anti-M13 antibody (1:10,000) was added. After washing, bound rabbit antibody was visualized and quantitated by reaction with horseradish peroxidase conjugated goat anti-rabbit antibody (1:1000) and subsequent development with the chromogenic substrate O- phenylenediamine.
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO

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Abstract

Cette invention se rapporte à de nouvelles molécules biologiquement actives qui se lient au récepteur de thrombine et l'inhibent. Les molécules de cette invention se caractérisent par une partie qui se lie au domaine 'analogue à l'hirudine' du récepteur de thrombine (aminoacides 52-69). Ces antagonistes de récepteur de thrombine se caractérisent en outre par leur aptitude à inhiber l'agrégation plaquettaire induite par thrombine sans affecter l'activité catalytique de la thrombine. Plus particulièrement, l'antagoniste de récepteur de thrombine de cette invention répond à la formule: Y-A1-W-X1-X2-X3-X4-A2-Z; Y représentant hydrogène, un peptide contenant de 1 à 10 aminoacides, identiques ou différents, ou une chaîne acyle ou alkyle contenant de 1 à 30 atomes faisant office de squelette; A1 et A2 étant identiques ou différents, et étant chacun choisis dans le groupe composé d'un aminoacide à charge positive et d'une chaîne acyle ou alkyle contenant de 1 à 10 atomes faisant office de squelette et un groupe latéral à charge positive; W et chaque X représentent n'importe quel aminoacide, identique ou différent; et Z représente OH, un peptide contenant 1 à 11 aminoacides, identiques ou différents, ou une chaîne acyle ou alkyle contenant 1 à 33 atomes faisant office de squelette, à condition qu'au moins un élément parmi W, X1 et X2 ne représente pas un aminoacide de base; ledit antagoniste étant éventuellement cyclisé par une liaison covalente entre Y et Z. Cette invention se rapporte également à des compositions et à procédés permettant l'utilisation de ces molécules à des fins thérapeutiques et prophylactiques.
EP93907162A 1992-03-02 1993-03-02 Antagonistes de recepteur de thrombine Withdrawn EP0632829A1 (fr)

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